Abstract
Primary structural studies have been carried out on the light chain of a homogeneous rabbit antibody to Group C streptococcal carbohydrate. Approximately 20 g of this antibody were obtained by multiple exchange transfusions at the peak of the antibody response. The isolated antibody was homogeneous by several antigenic and chemical criteria. The light chain was isolated and modified, and then digested with alpha-chymotrypsin or thermolysin. The resulting peptides were isolated by gel filtration, paper electrophoresis, and paper chromatography. The amino acid sequences of these peptides were determined by Edman degradation plus dansylation. This supplied sufficient information to assign approximately 90 percent of the residues in the chain. The destruction of tyrosine during acid hydrolysis of peptides which had been eluted from a paper chromatogram was investigated. This destruction is due to inpurities in the paper which contaminate the peptides. Prevention of such destruction can be achieved by predevelopment of the paper with 1 N NH4OH prior to paper chromatography.
Highlights
20 g of this antibody were obtained by multiple exchange transfusions at the peak of the antibody response
The resulting peptides were isolated by gel filtration, paper electrophoresis, and paper chromatography
The amino acid sequences of these peptides were determined by Edman degradation plus dansylation
Summary
Materials a-Chymotrypsin and diisopropylphosphorofluoridate-treated carboxgpeptidases A and B were purchased from Worthington. Bioche-&ical Corp.; thermolysin from Calbiochem; aminopeptidase M from Rohm and Haas. Germany; ethylenimine from Eastman Kodak Co ‘. Dithiothreitol from- Caibiochem; phenylisothiocyanate, pyrizne, trifluoroacetic acid, and dithioerythritol from Pierce Chemical Co., Rockford, Ill.; DEAEcellulose from the Whatman Corp.; iodoacetamide from Sigma. Corp., St. Louis, L-tyrosylglycine from Schwarz/Mann, and Whatman chromatofrranhv naner from W and R Balston Ltd., England. Purijication of Homogeneous Antibody-Rabbit 4135 was immunized with streptococcal Group C vaccine as previously described [2] and a large volume of immune plasma was collected by exchange transfusions as described by Greenblatt el al. The antibody levels were measured and microzone electrophoresis was carried out as previously described [2]
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